Abstract This application is in response to Recovery Act Limited Competition: NIH Director's Opportunity for Research in Five Thematic Areas (RC4). In particular we are responding to (Theme 1) Applying Genomics and Other High Throughput Technologies and (Theme 5) Reinvigorating the Biomedical Research Community. Modern molecular medicine, propelled by the human genome project, has been exceptionally successful at identifying specific mutations that contribute to disease. But the critical paths by which mutations lead to pathology remain obscure in most cases. This crippling disconnect between genetic insight and effective therapeutic intervention frustrates the biomedical research community and disillusions the general public. The problem is so pervasive because mutations rarely alter protein activities by simple, direct effects on active sites. Based on the cases characterized to date, mutations most commonly disrupt a protein's ability to fold efficiently and/or perturb the interactions with other proteins that are essential for its function. Dramatic examples include mutations in the chloride channel responsible for cystic fibrosis and the oncogenically activated tyrosine kinases that drive many human cancers. In the former, a minor problem in the folding of CFTR leads to excessive degradation of what could otherwise be a functional channel. In the latter, oncogenic mutations of many different proteins often leads to aberrant, promiscuous interactions with other proteins and an extreme dependence on the Hsp90-based chaperone machinery, a prominent feature now being targeted in the development of anticancer Hsp90 inhibitors. The research community lacks a resource to study these fundamental issues on anything approaching the global scale that is needed. In direct response to the opportunity afforded by this RC4 "Recovery Act Limited "Competition" we aim to provide this resource by "Applying Genomics and Other High Throughput Technologies" (Theme 1), thereby "Reinvigorating the Biomedical Research Community" (Theme 5). We will create the first comprehensive library of disease-associated mutant genes, determine the effect of each mutation on interaction with key components of the cellular chaperone machinery, map genome-wide interactions with other proteins. Open Reading Frames (ORFs) encoding ~10,000 different disease causing alleles will be generated using a recombinational cloning system and verified by "next gen" sequencing via the high-throughput pipeline operational in our center. Verified mutant and wild type ORFs will be functionally characterized using distinct yet complementary high-throughput approaches that are already "on-line" in our labs. First, the biochemical interaction of disease alleles with Hsp90 and its co-chaperones will be assessed quantitatively. Second, alleles will be expressed in yeast to probe for genetic interactions with any of ~18,500 human genes in our ORFeome collection using a high-quality 2-hybrid pipeline. Protein-protein interactions that are abolished by mutation or which only the mutant protein can undergo will be identified at a scale that has never before been achieved. Convincing validation will be obtained for this unique resource and a transformative view of how mutations cause disease will be enabled. PUBLIC HEALTH RELEVANCE: Relevance to Public Health Modern genomic technologies have identified a large number of gene mutations that cause a variety of human diseases, however, in most cases, the way genetic mutations actually cause disease in humans is unclear, largely because mutations alter protein interactions rather than directly disrupting protein function. This problem has led to a major roadblock in using genetic information to develop effective new treatments. This project will create a unique resource of ~10,000 mutant genes, selected on the basis of their involvement in over 2,500 different human genetic diseases to examine how mutations can alter protein-protein interactions in cells on a genome-wide scale. The never before achievable insights will dramatically improve the translation of current genetic knowledge to actual improvements in treatment and outcome for patients.